Plant Diversity 38 (2016) 253e258

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Plant Diversity
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http://journal.kib.ac.cn

Reproductive biology of Magnolia sinica (Magnoliaecea), a threatened

species with extremely small populations in Yunnan, China
Ye Chen a, b, Gao Chen a, Jing Yang a, *, Weibang Sun a, **
a
Kunming Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan, China
b
University of Chinese Academy of Sciences, 100049 Beijing, China

a r t i c l e i n f o a b s t r a c t

Article history: Magnolia sinica is one of the most threatened trees endemic to Southeast Yunnan. Based on our in-
Received 27 May 2016 vestigations, only 52 individuals and eight populations are found in the wild. M. sinica has been cate-
Accepted 7 September 2016 gorized as Critically Endangered on the IUCN Red List and identified as a “Plant Species with Extremely
Available online 13 September 2016
Small Populations (PSESP)”. Its fruit/seed set is very low and seedlings are rarely found in the wild. It is
(Editor: Zhekun Zhou) hypothesized that it may encounter obstacles to reproductive success. This study, therefore, focuses on
its reproductive biology, knowledge of which is essential for effective conservation. Flowers of this
Keywords: species are protogynous and nocturnal, and possess a two-day rhythm of sexual presentation. For the
Magnolia sinica first night of anthesis, the flowers are in the pistillate stage during which tepals open at dusk and close
PSESP approx. 1 h later (except for the open outer ones). They remain closed until the next afternoon, when
Flowering process flowers, now in the staminate stage, re-open and remain so until the tepals drop. Nocturnal beetles enter
Pollination into the flowers and remain trapped throughout the night as the flower closes, during which time they
Seed dispersal feed on tepals. Pollen-gathering bees are found to visit the re-opened flowers and the beetles are
Germination
released during this stage. Two species of Pleocomidae and Curculionidae beetles appear to be effective
pollinators.
M. sinica is a self-compatible, pollinator-dependent species, and its fruit/seed set can be significantly
increased by hand-pollination. No functional seed dispersers have been found in its extant natural
habitats. These findings suggest that it may face both pollination and seed disperser insufficiencies in its
current fragmented habitats, which may account for its low regeneration. Here we propose conservation
strategies based on our findings.
Copyright © 2016 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by
Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Magnolia sinica (Law) Noot. (Magnoliaceae), a rare tree species
endemic to Southeast Yunnan, China, was first described as Man-
glietiastrum sinicum Law in 1979 (Law, 1979). Most Chinese bota-
nists often call this species M. sinicum and also use its common
name huagaimu (Sun et al., 2012; Wang et al., 2016). Its other
synonyms are Manglietia sinica (Chen and Nooteboom, 1993) and
Pachylarnax sinica (Xia et al., 2008). Because the species has been
referred to as M. sinica internationally, M. sinica is also used in the
present study.
* Corresponding author.
** Corresponding author.
E-mail addresses: yangjing@mail.kib.ac.cn (J. Yang), wbsun@mail.kib.ac.cn
(W. Sun).
Peer review under responsibility of Editorial Office of Plant Diversity.
M. sinica distributes in south subtropical monsoon broadleaved
evergreen forests and scatters at altitudes between 1339 and
1707 m (Wang et al., 2016). Recent anthropogenic activities,
including deforestation for commercial cultivation (e.g. Amomum
tsao-ko Crevost et Lem. and Cunninghamia lanceolata) and habitat
destruction, have lead both to the reduction of its population size
and to serious habitat fragmentation. This species is categorized as
Critically Endangered on the IUCN Red List (Cicuzza et al., 2007;
Rivers et al., 2016) and has been identified as a “Plant Species
with Extremely Small Populations (PSESP)” (State Forestry
Administration of China, 2012; Ren et al., 2012; Ma et al., 2013;
Volis, 2016) for priority conservation in China. It has also been
targeted as one of the 20 species approved by the Yunnan gov-
ernment for urgent rescue action before 2015 (Wang et al., 2016). In
the field, a total of 52 individuals of M. sinica were isolated in eight
isolated populations. Of these 52 individuals, nine are young trees

http://dx.doi.org/10.1016/j.pld.2016.09.003
2468-2659/Copyright © 2016 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This
is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
254 Y. Chen et al. / Plant Diversity 38 (2016) 253e258
with a DBH (diameter at breast height) of less than 22.5 cm (Wang
et al., 2016).
Although M. sinica flowers well, its fruit/seed set is low and
seedlings are rarely found in the wild. Conservation via ex situ
cultivation in gardens and reinforcement/reintroduction in the wild
have been conducted by Kunming Botanical Garden (Sun, 2013).
However, the rarity of seedling/saplings in the wild limits the po-
tential for the natural regeneration of this species. Therefore,
despite ex situ and in situ conservation trials, effective conservation
measures based on scientific studies are imperative.
Knowledge of reproductive biology is essential for the effective
protection of endangered plants, especially for species with small
populations (Spira, 2001; Evans et al., 2004; Xiao and Xu, 2006).
Successful reproduction is crucial in maintaining a viable popula-
tion size, which is of critical concern to highly endangered taxa
facing extinction (Pandit and Babu, 2003; Gong et al., 2014). In
species relying on seeds to recruit new individuals into populations,
population viability may be closely related to seed dynamics and
conservation measures may depend on understanding the factors
that limit seed production (Pavlik et al., 1993; Zhao and Sun, 2009)
and dispersal. When seed production is mediated by pollinators, it
can be influenced by pollinator abundance or behavior
(Bierzychudek, 1981; Larson and Barrett, 2000). It may be limited
by pollen, because self-pollen may cause reduction in seed pro-
duction through inbreeding depression (Bosch and Waser, 1999;
Brown and Kephart, 1999; Zhao and Sun, 2009). Seed production
in particular, as a seed source for offspring, may directly affect seed
dispersal. Seed dispersal may also be influenced by disperser
abundance or behavior, if seed dispersal is mediated by dispersers.
Studies on the reproductive biology of threatened species with
low reproductive capabilities have been the focus of recent research
and have become an important aspect of conservation manage-
ment. Despite extensive attention and critical conservation status,
little information is available on the reproduction of M. sinica. The
current study was undertaken to gain knowledge of the repro-
ductive characteristics of M. sinica and aims to address the
following issues: (1) its floral biology, especially the flowering
process; (2) its pollination biology and the role of pollinators in fruit
set; (3) its breeding system; (4) the characteristics of seed dispersal
and seed germination of the species in the natural habitats.
1. Methods and materials
1.1. Study sites
The pollination biology of M. sinica was investigated from 2014
to 2015 in Jingping County of Honghe Hani-Yi Autonomous Pre-
fecture. The population there comprises four individuals. One is
located beside a mountain trail in Zhongliang village and the other
three are located in the thick forest on Luoguoping Mountain.
Reproductively mature individuals of M. sinica are usually tall trees.
The tree in Zhongliang village was chosen as the experimental
subject because of its convenience and easy access, and a 17 m high
stand was built around it for experiments. All observations and
experiments on the reproduction of the species were carried out on
this tree, in the middle of March when M. sinica began flowering.
The seed dispersal experiment was conducted in November
2014 in a natural habit of M. sinica in Chinese fir seed orchards
(label DLS-T), located in Dalishu Township of Maguan County in
Wenshan Zhuang-Miao Autonomous Prefecture.
Further seed germination experiments were conducted in
November 2014, at three natural localities: DLS-T, Zhongliang
village of Jingping County in Honghe Hani-Yi Autonomous Prefec-
ture (label ZL-V), and Miechang Township of Maguan County in
Wenshan Zhuang-Miao Autonomous Prefecture (two plots, label
MC-T-1 and MC-T-2).
1.2. Pollen viability and stigma receptivity
The examination of pollen viability and stigma receptivity was
conducted on three flowers per day over five consecutive days at
full-bloom stage in both 2014 and 2015.
Pollen viability was examined using the MTT method (Dafni,
1992). After pollen has been mixed with MTT solution and left for
10 min, viable pollen will turn dark purple in color whereas inviable
pollen will turn tawny-yellow or remain unchanged. Pollen was
taken from anthers at different flowering stages and mixed evenly
with 1‰ MTT on two slides. The numbers of viable and inviable
pollen grains were counted over three separate views per slide,
using a light microscope.
Stigma receptivity was examined by the benzidine-hydrogen
peroxide method (Dafni, 1992). After soaking in a benzidine-
hydrogen peroxide solution, a receptive stigma will show peroxi-
dase activity and turn blue with a mass of bubbles surrounding it.
The depth of the blue color indicates the intensity of receptivity. At
different flowering stages, two stigmas per flower were picked and
assessed for mucus secretion. They were then soaked in the
benzidine-hydrogen peroxide solution. The changes of color and
occurrences of bubbles were observed by eye.
1.3. Flowering dynamics
Based on primary observations, flower buds that are about to
open can easily be recognized by the softness of their bud tips and
their stronger fragrance, and some mature flower buds will open
simultaneously at the same time every dusk over the flowering
duration of the tree. In every flowering-season over two consecu-
tive years, five flowers were labeled and their flowering stages were
observed continuously until the tepals wilted.
1.4. Observation of floral visitors
Observations of floral visitors were carried out from 8:00 AM to
22:00 PM over four consecutive days, spreading across two flow-
ering seasons: March 2014 and April 2015. During observations, five
or six flowers were randomly labeled per day. Visitors to these
flowers were caught using tweezers and kept in 70% ethyl alcohol
for subsequent identification in the laboratory.
1.5. Breeding system
To evaluate the breeding system of M. sinica, flower buds that
would definitely blossom at dusk were selected randomly, marked
and caged with sulfate paper bags (30
25 cm) every day in the
morning at the full-bloom stage in both 2014 and 2015. Five
pollination treatments were assigned at the moment when the
flowers started to open. Autonomous self-pollination (label SPON,
n ¼ 12) was tested by bagging flowers to exclude visitors. Self-
compatibility was tested by bagging emasculated flowers and
transferring their own pollen by hand (label SELF-S, n ¼ 22) and
bagging emasculated flowers and being hand-pollinated with
pollen from other flowers at the same tree (label SELF-D, n ¼ 62). To
test for xenogamy (label CROSS, n ¼ 47), flowers were treated
similarly but were hand-pollinated with pollen from other trees.
Control flowers were unmanipulated (label OPEN, n ¼ 150).
The marked flowers were harvested in early November, when
fruits were collected to assess fruit and seed set.
The self-compatibility index (SCI) was used to determine the
breeding system of M. sinica and was obtained as mean percentage
 Y. Chen et al. / Plant Diversity 38 (2016) 253e258
fruit set from hand self-pollination over that from hand cross-
pollination. Species with SCI ratios 0.2 are considered to be self-
incompatible, otherwise, they are considered to be self-
compatible (Jhumur et al., 2008).
1.6. Seed dispersal and seed germination in the wild
Field trials were conducted to determine the seed-dispersal
strategy of M. sinica. A total of 900 seeds were assigned to the
following six treatments (five replicates of 30 seeds each): seeds
with red arils were distributed: (a) on a dehiscent fruit shell which
was bound above the ground on a branch, (b) in a circle (diameter,
ca. 1 m) on the ground. Seeds with the arils removed, and half seeds
with red arils and half seeds with the aril removed were arranged
the same ways. In the treatments where seeds were placed on the
ground, every seed was labeled with a numbered plastic tag
(1 cm
3 cm) attached by a thin stainless-steel wire (diameter, ca.
0.2 mm) 10 cm long, similar to the procedures reported by Xiao
et al. (2006). This seed-tagging method allowed us to follow the
exact fate of the seed over time. The seed-carrying behavior of
rodents and birds was monitored continuously by infrared cameras.
During seven consecutive days, numbers of seeds remaining were
recorded, and, during each visit, seeds that had been removed were
retrieved from around seeds stations (diameter of searching area,
ca. 10e15 m) at dusk.
To test the effects of removing red arils from seeds on germi-
nation rates, germination assays were performed on a total of 800
seeds, 400 seeds of which the red arils were removed. A total of 100
seeds with red arils and 100 seeds with arils removed were paired
and sowed in ZL-V. Other seeds were arranged in the same way in
MC-T-1, MC-T-2 and DLS-T. All seeds were buried in a circle
(diameter, ca. 1 m) under the surface soil at a depth of 6 cm. Con-
ditions of seed germination were recorded, and the germination
rates of seeds with red arils and seeds with arils removed were
compared in 2015.
2. Results
2.1. Flowering process
Two consecutive years of observation of M. sinica at the exper-
imental site showed that flowering started around mid-March and
lasted until the middle of April. The peak flowering time was from
late March until early April. The terminal, bisexual flowers opened
and closed in a two-day rhythm and the flowering period for a
single flower was three to four days. Based on data gathered from
pollen viability and stigma receptivity experiments described
below, five different flowering stages were distinguished to clarify
floral processes and pollination.
Before a flower opened, several whorls of green bracts detached,
and, after the last whorl of a red bract detached, the pink-purple
tepals were exposed. The pre-pistillate stage commenced in the
morning (ca. 8:00 AM) and ended at dusk (ca. 18:30 PM) (Fig. 1A).
During this stage, the flower bud became soft and emitted a strong
fragrance, and this was the indication that the tepals would open
soon and the anthesis of this flower would be initiated. The
pistillate stage commenced at ca. 18:30 to 19:00 PM and continued
until ca. 20:30 PM. The tepals started to separate and moved out-
wards to completely open within 10e15 min (Fig. 1BeC), revealing
the gynoecium. The green stigmas were brilliant with viscous, non-
odorous exudates, and the anthers were closed tightly at the basal
part of the torus. Beetle visitors were found present and active in
the flower. Tepals of the flower in the pistillate stage started closing
approximately 1 h later (ca. 20:10 PM) within 15e20 min (Fig. 1D),
indicating that the pistillate stage ended and the pre-staminate
255
stage initiated. The pre-staminate stage continued throughout the
whole night until ca. 12: 00 AM of the next day, when the anthers
started dehiscencing introrsely. The re-closed flower in this stage
could be easily distinguished from the pre-pistillate flower by the
extended outer tepals (Fig. 1E). The staminate stage lasted from ca.
12:00 AM to 17:30 PM of the second afternoon. The flower began to
re-open approximately 3.5 h (ca. 15:30 PM) after the commence-
ment of the staminate stage and was completely open after
10e15 min (Fig. 1F). Stigmas gradually turned yellowish without
obvious exudates. The wilting stage commenced at ca. 17:30 PM
and lasted for 1e2 days until the stigmas turned black and wilted,
and anthers and tepals gradually detached from the receptacle.
2.2. Pollen viability and stigma receptivity
The examination of pollen viability revealed that during the pre-
pistillate stage and the pistillate stage, pollen was immature and
inviable. Some pollen began to show viability (42.03 ± 3.98%) the
following morning (about 8:30 AM) and the overall pollen viability
increased to a peak (93.93 ± 0.71%) 30 min after the anthers
dehisced at about 12:00 AM. After that, pollen viability declined to
80% at the beginning of the wilting stage when the anthers started
to fall from the flower.
Stigmas receptivity was weak during the pre-pistillate stage,
though it increased up until the opening of the flowers at the
pistillate stage. Stigma receptivity then declined until the end of
staminate stage.
2.3. Observation of flower visitors
Visitors observed during flowering period were unidentified
species of beetles belonging to the taxa Pleocomidae, Cetoniidae,
Curculionidae, Geotrupidae, Carabidae, Elateridae and Scar-
abaeidae and bees identified as Apis mellifera (Apidae). Based on
observations made over two years, the beetles and bees showed
different visitation patterns.
Coleoptera beetles were observed to be relatively abundant
between about 18:30 and 20:30 PM. The beetles entered the
flowers either from the tip of the tepals or across the base of tepals
(Fig. 1GeH) and became trapped inside in the floral chamber when
the tepals re-closed tightly in the pre-staminate stage and the
staminate stage. Inside the re-closed flowers, beetles continued
actively moving and feeding on tepal tissue. Upon the re-opening of
the flowers, the beetles were finally released (Fig. 1IeJ). During the
visitation, only two species of Pleocomidae (Fig. 1KeL) and Cur-
culionidae beetles were observed to enter and leave the chamber
carrying pollen grains, suggesting that these two beetles might be
effective pollinators, while the role of other species remained un-
clear. Beetle excrement was sometimes found filling the interior
tepals of flowers that had been visited (Fig. 1M), although the
stigmas of these flowers suffered no damage. A. mellifera visited
only when the flowers re-opened with peak abundance at
15:30e16:30 PM, and with a visiting frequency of 28.3 ± 1.47 h1.
As the bees only occurred at the staminate stage to gather pollen
grains, they might not be the effective pollinators. Some flowers
had their androecia severely damaged (or detached) as a result of
vigorous pollen gathering by A. mellifera (Fig. 1N).
2.4. Breeding system
The mean percentage fruit set for the five pollination treatments
are given in Fig. 2A. The fruit set resulting from natural pollination
(OPEN) was 22 ± 3%. Bagging-flowers without emasculation (SPON)
yielded no fruits, indicating that autonomous self-pollination was
not possible in this species. The hand-pollinated within flower
256 Y. Chen et al. / Plant Diversity 38 (2016) 253e258

Fig. 1. Flowering process, insects visitors and fruits of M. sinica. (A) A bud tip at the pre-pistillate stage; (B)e(C) Tepals separating and moving outwords to a fully open at the
pistillate stage; (D) Tepals re-closing at the end of the pistillate stage; (E) Tepals except for the outer ones remaining closed during the pre-staminate stage; (F) The tepals completely
re-opening at the staminate stage; (G)e(H) Beetles entering the open chamber at the pistillate stage. (I)e(J) Beetles leaving the re-opening chamber at the staminate stage; (K)e(L)
Beetles (Pleocomidae) visiting the open flower, touching the exudate-secreting stigma; (M) Tepals partly eaten on the inside by visiting beetles. (N) Bees (A. mellifera) visiting a re-
opening flower during the staminate stage; (O) Young fruit; (P) Mature fruit with red aril on seeds.

Fig. 2. Fruit sets and seed germination rates of M. sinica (A) Fruit sets (mean percentage and standard error) with different pollination treatments; (B) Seed germination rates in
different plots.

(SELF-S) mean percentage fruit set was 63.64 ± 0.00%, which was
marginally higher than that under hand-pollinated within the plant
treatments (SELF-D) (58.15 ± 8.06%). The fruit set resulting from
outcrossed hand-pollinations was recorded as 69.04 ± 4.84%.
ANOVAs manifested differences between hand-pollinations and
pollination under controlled conditions were significant (F1,
8 ¼ 6.47, p ¼ 0.003). Nevertheless, fruit set under hand-pollination
within the same plant was not significantly different from that
under outcrossed hand-pollinations (F1, 6 ¼ 0.33, p ¼ 0.586). The SCI
ratio was calculated as 0.86. This showed that M. sinica was mostly
self-compatible. The seed set from hand-pollination was
31.77 ± 1.10%, but the seed set from natural pollination was
 Y. Chen et al. / Plant Diversity 38 (2016) 253e258
16.97 ± 0.19%. The difference between the two treatments was
significant (F27, 39 ¼ 8.51, p ¼ 0.004).
2.5. Seed dispersal and seed germination in the wild
Our study did not record birds, rodents or other animals taking
M. sinica seeds, either with or without red arils, in the wild. This
may indicate that the experimental site lacked effective animal
populations for M. sinica seed dispersal, or that frequencies of an-
imal vector visitation were rather low.
2.6. Seed germination
The germination rates of the seeds with red arils and seeds with
arils removed as well as the seed germination rates in ZL-V, MC-T-1,
MC-T-2 and DLS-T are shown in Fig. 2B. Because of inappropriate
sowing-plot (a path to the farmland) in ZL-V, the seed germination
rate here was zero and was excluded from the following analysis. In
addition, the differences in the seed germination rates between
MC-T-1, MC-T-2 and DLS-T were small in ANOVAs (F2, 3 ¼ 1.09,
p ¼ 0.441). The germination rate of seeds with red arils sowed in
MC-T-1, MC-T-2 and DLS-T was calculated as 9 ± 2.13%, much lower
than that of seeds with the aril removed (16.67 ± 3.82%). However,
ANOVAs suggested that the differences in germination rates be-
tween seeds with red arils and seeds with arils removed were not
significant (F1, 4 ¼ 0.68, p ¼ 0.455). Thus, red arils did not greatly
affect seed germination in this species.
3. Discussion
Based on the relatively high SCI and fruit sets resulting from
SELF-S and SELF-D treatments, we concluded that M. sinica was
self-compatible. Significant differences in fruit sets between polli-
nator exclusion (SPON) and control treatment (OPEN) indicated
that pollinators were necessary for the reproductive success of
M. sinica. In addition, the significant differences between hand-
pollinations and pollination under controlled conditions further
suggested that hand-pollinations could increase fruit set and this
species was pollinator dependent. Thus, we hypothesized that
M. sinica was encountering pollination limitation. Pollination lim-
itation, including pollinator limitation and pollen limitation, which
is of great concern for seed production, may occur in small, isolated
populations (Huang and Guo, 2002). Using hand-pollination, the
quantity and quality of pollen received have been shown to be
important factors affecting the seed production in certain species
(Zimmerman and Pyke, 1988; Johnston, 1991). Limited pollen flow
resulting from pollination limitation in the population may
decrease the effective population size and reduce progeny fitness
(Barrett and Kohn, 1991; Washitani, 1996). Individuals of M. sinica
are known to be only sporadically distributed through its small and
fragmented range. Pollen resources for pollination might therefore
be limited.
M. sinica was found to be protogynous and displayed a two-day
rhythm of sexual presentation, with a few hours (approx. 21 h,
during which time stigmas were receptive) being of critical
importance for successful fruit production. Protogyny is a charac-
teristic of beetle-pollinated plants (Bertin and Newman, 1993;
Momose et al., 1998; Azuma et al., 1999) and many species in the
Magnoliaceae are protogynous and beetle-pollinated (Dieringer
and Espinosa, 1994; Gottsberger et al., 2012; Dieringer et al.,
2015). Furthermore, for the eight species of temperate Magnolia
studied by Thien (1974), flowers frequently exhibited diurnal petal
movements over 2 d of anthesis, and were visited by several taxa of
Coleoptera. However, compared to these species, M. sinica appeared
to possess a much shorter opening period (1e1.5 h) for nocturnal
257
beetle visits. In this system, the re-closed tepals formed wide floral
chambers and the plants forming such chambers are mostly beetle-
pollinated (Dieringer et al., 2015). The floral chambers have been
suggested to induce long beetle visits by providing shelter, food and
sometimes potential mating places (Dieringer et al., 2015). The
beetles may stay inside for a longer period to be protected from
predation and to some extent from adverse ambient conditions,
thus increasing potential pollination for such species. We found
that the tepals re-opened and exposed dehiscent anthers on the
second afternoon, releasing trapped beetles. Two species of Pleo-
comidae and Curculionidae beetles were observed to enter and
leave the chamber carrying pollen grains, indicating that M. sinica
was certainly beetle-pollinated and the two species of Pleocomidae
and Curculionidae beetles were effective pollinators of M. sinica.
Mature fruits of M. sinica finally split into an irregular star-like
structure, exposing the seeds, which have red fleshy arils.
(Fig. 1OeP). This indicates that the red color may act as an attrac-
tant to potential dispersal by birds (Duan et al., 2014). However, our
field experiments did not observe animals, nor consequently seed
dispersers in the typically fragmented habitats of M. sinica.
Anthropogenic factors can have cascading effects on seed dispersal
(Hamann and Curio, 1999; Meehan et al., 2002; Oppel, 2010).
Habitat fragmentation may reduce the abundance of a suite of
frugivorous fauna, which in turn reduces seed dispersal of the plant
species (Moran et al., 2009). In modern times, as has occurred in
Southeast Asia (Bennett et al., 2000; Corlett, 2007; Harrison, 2011),
increased human population and a switch to modern technology,
for example firearms and nylon mist nets, have lead to increased
hunting pressure and the consequent extinction of many species
(Zhang et al., 2014).
Natural habitats for M. sinica have been greatly altered by
reforestation of C. lanceolata. Also, since the ornamental value of
M. sinica was recognized in the early 1980s, local villagers have
been collecting its seeds every fruit season, which they then sell at a
high price in the market. Our field investigations found that at least
some 10,000 cultivated seedlings and saplings have been cultivated
in nurseries, which may contribute to ex situ conservation of
germplasm resources of M. sinica. But the massive collection of
seeds will also aggravate the lack of seeds dispersing in the wild
and make it more difficult for population regeneration.
Therefore, conservation strategies for this PSESP species should
focus on the following aspects. First, in order to diminish pollen
limitation, saplings should be recruited into the extant populations
to increase the population density and provide more pollen re-
sources. Second, small nature reserves should be established to
control the damage to natural habitats of M. sinica and promote the
recovery of its original habitats, so that it can enhance the polli-
nator/disperser assemblage. Though the seeds of M. sinica can
germinate in the wild, our investigations rarely found seedlings.
Thus, seedling morphogenesis may be another limitation to the
recruitment of M. sinica and should also be given attention to in the
future.
Acknowledgements
This study was supported financially by the NSFC-Yunnan joint
fund to support key projects to W.B. Sun (Grant no. U1302262) and
the Young Academic and Technical Leader Raising Foundation of
Yunnan Province to G. Chen (2015HB091). The authors thank
Guoyun Li, Zhiyong Yu and appreciate the cooperation of the
Honghe and Wenshan forestry authorities. The first author wishes
particularly to thank Rongli Liao and Bin Wang for helping carry out
the experiments and Lingzeng Meng for the identification of the
insects.
258 Y. Chen et al. / Plant Diversity 38 (2016) 253e258

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